Laboratory Testing for Developmental Delay, Intellectual Disability, and Autism Spectrum Disorder

Medical Experts

Contributor

Bayrak-Toydemir

Pinar Bayrak-Toydemir, MD, PhD, FACMG

Professor of Pathology (Clinical), University of Utah

Medical Director, Molecular Genetics and Genomics, ARUP Laboratories

Contributor

Bosworth

Michelle Bosworth, MS, LCGC

Genetic Counselor, ARUP Laboratories

Contributor

LaGrave

Danielle LaGrave, MS, LCGC

Faculty, Genetic Counseling Graduate Program, Human Genetics, University of Utah

Genetic Counselor, ARUP Laboratories

Contributor

Longo

Nicola Longo, MD, PhD

Professor, Pediatrics; Adjunct Professor of Pathology (Clinical), University of Utah

Chief, Medical Genetics Division; Medical Director, Biochemical Genetics and Newborn Screening, ARUP Laboratories

Contributor

Mao

Rong Mao, MD, FACMG

Professor of Pathology (Clinical), and Co-Director of Laboratory Genetics and Genomics Fellowship, University of Utah

Medical Director, Molecular Genetics and Genomics, ARUP Laboratories

Contributor

Pasquali

Marzia Pasquali, PhD, FACMG

Professor of Pathology, Adjunct Professor of Pediatrics, University of Utah School of Medicine

Section Chief, Biochemical Genetics; Medical Director, Biochemical Genetics and Newborn Screening, ARUP Laboratories

Contributor

Rudd

Katie Rudd, PhD, FACMG

Medical Director, Cytogenetics and Genomic Microarray, ARUP Laboratories

Developmental delay (DD) is defined as any significant lag in a child's physical, cognitive, emotional, or social maturity. Intellectual disability (ID) is characterized by broad impairment in cognitive and adaptive functioning, typically with an intelligence quotient (IQ) <70, with onset before 18 years of age. A global DD diagnosis often precedes a diagnosis of ID because neither cognitive skill nor IQ can be reliably assessed before age 6. Those with severe DD diagnosed before age 6 are most likely to develop ID.^,

Autism spectrum disorder (ASD) represents a neurodevelopmental continuum with varying degrees of social impairment, communication limitations, and repetitive or restrictive behaviors. Routine pediatric screening may identify a child with global delay and spur investigation of the underlying etiology. Laboratory tests are not used to diagnose ASD but are instead used to investigate its etiology and identify if other medical conditions are present.

Chromosomal microarray (CMA, also referred to as genomic microarray) is a recommended first-line test for DD/ID or ASD of unknown etiology.^,^, Multiple groups have made recommendations regarding additional testing for fragile X syndrome (FXS), inborn errors of metabolism (IEMs), X-linked intellectual disability, MECP2-related disorders, PTEN-related disorders, and/or chromosome analysis, depending on clinical findings and family history.^,^,^, The American College of Medical Genetics and Genomics (ACMG) recommends consideration of exome or genome sequencing as a first- or second-tier test for the evaluation of pediatric-onset DD/ID.

Refer to the associated testing algorithms, Testing for Genetic Syndromes Related to Developmental Delay and Intellectual Disability and Testing for Genetic Syndromes Related to Autism Spectrum Disorder, for suggested tiered testing approaches based on clinical presentations.

Quick Answers for Clinicians

How are whole genome sequencing and whole exome sequencing used in the evaluation of developmental delay, intellectual disability, and autism spectrum disorder?

Although the American College of Medical Genetics and Genomics (ACMG ) now strongly recommends consideration of whole genome sequencing (WGS) or whole exome sequencing (WES) in the evaluation of developmental delay (DD) and intellectual disability (ID), these techniques are not universally used or available. Technology continues to evolve and costs have continued to decline, factors which have made adoption of WGS and WES more feasible and have allowed more practices to use these methods as first- or second-line tests. Both WGS and WES have demonstrated clinical value in DD and ID, including for confirmation of diagnoses and informing management. Therefore, it is likely that WES and WGS will be used more often in the evaluation of these conditions. In autism spectrum disorder (ASD), genetic testing identifies the etiology of symptoms and provides helpful information to patients and their families. Additionally, in ASD, the American Academy of Pediatrics (AAP) states that genetic testing such as WES may be offered to patients and their families as part of the laboratory workup.

What role does chromosome analysis (standard karyotyping) play in the workup for developmental delay/intellectual disability or autism spectrum disorder?

Chromosomal microarray (CMA) has replaced karyotype analysis as the first laboratory test for developmental delay (DD), intellectual disability (ID), and autism spectrum disorders (ASDs) of unknown etiology.^,^, CMA is also now the first-line test for patients with multiple congenital anomalies (MCAs). Although chromosome analysis was historically the first-line test for patients with DD/ID, it is currently only recommended as a first-line test when features are consistent with aneuploidy (eg, Down syndrome, trisomy 13, trisomy 18, Klinefelter syndrome, or Turner syndrome). Chromosome analysis is also indicated when there is a family history of chromosome rearrangement or multiple miscarriages because it can detect balanced chromosomal abnormalities, which CMA does not detect.^, To evaluate a patient with MCAs/DD/ID/ASD who is at risk for aneuploidy, chromosome analysis with reflex to CMA is a good choice.

Is there a role for FISH analysisin the evaluation of developmental delay, intellectual disability, or autism spectrum disorder?

Fluorescence in situ hybridization (FISH) probes assess a specific genomic region, whereas chromosomal microarray (CMA) detects copy number variants (CNVs) across the entire genome.^, FISH is less expensive than CMA, but if FISH analysis does not confirm the suspected diagnosis, additional testing must be performed. CMA can identify CNVs that are too small to identify via FISH, and smaller duplications detected by CMA may not be visible by FISH. Metaphase FISH shows the genomic location of a duplicated region, but CMA does not. Thus, FISH metaphase analysis is used to assess cryptic balanced rearrangements in relatives of a proband with an unbalanced rearrangement.

What are the expected diagnostic yields for laboratory tests used in the evaluation of developmental delay, intellectual disability, and autism spectrum disorder?

The diagnostic yields of different laboratory tests vary for developmental delay (DD)/intellectual disability (ID) and autism spectrum disorder (ASD). For more information, refer to the Diagnostic Yields of Genetic Testing table.

How does the laboratory workup for multiple congenital anomalies compare with the workup for developmental delay and intellectual disability?

The laboratory workup for multiple congenital anomalies (MCAs) is very similar to the workup for developmental delay (DD) and intellectual disability (ID). The laboratory evaluation of MCAs includes chromosomal microarray (CMA) or whole exome/genome sequencing (WES/WGS) as first-line tests. If CMA is used as a first-line test, sequencing may be appropriate as a second-line test depending on findings. As with testing for DD and ID, additional testing for MCAs should be based on clinical presentation and judgment.

Indications for Testing

Laboratory evaluation of suspected DD, ID, or ASD should be considered for individuals with any of the following presentations^,:

Failure to meet developmental milestones
IQ <70 and difficulty performing daily living activities
Comorbidities (eg, dysmorphic features, congenital anomalies) that may guide testing

Determining Diagnosis for Care Planning

Clinical evaluation of DD/ID and ASD will guide the laboratory testing strategy and should include^,^,:

Medical history, including prenatal and birth histories
Developmental-behavioral history
Family history with at least a three-generation pedigree
Physical and neurologic exam with particular attention to dysmorphology (minor anomalies)

After the clinical evaluation, the judicious use of laboratory testing, imaging, and other techniques is recommended.^,^,^, If a specific condition is suspected, consider targeted testing. If the etiology of DD/ID/ASD is unknown, proceed with tiered testing based on which tests have the highest diagnostic yield in the patient population.

Laboratory Testing

The ACMG strongly recommends CMA or whole exome sequencing (WES) with fragile X testing, or whole genome sequencing (WGS) with consideration of microarray, in the evaluation of DD/ID of unknown etiology, due to the emerging evidence for therapeutic benefit and limited evidence for negative outcomes. When a specific disorder is suspected, a tiered approach is recommended.^, The American Academy of Pediatrics (AAP) recommends a similar stepwise approach in the evaluation of children with ID or global DD. The ACMG and AAP also recommend a tiered approach in the etiological evaluation of ASD.^, The logic behind this approach is that tests performed in higher (or earlier) tiers have a greater expected diagnostic yield, are less invasive, and provide better potential for intervention. The tiered approach also allows for customization to the clinical situation at hand.

First-Tier Evaluation

The first-tier evaluation for DD/ID includes a detailed clinical evaluation, as described in the Determining Diagnosis for Care Planning section. If a specific disorder is suspected, targeted testing should be performed with appropriate follow-up. If no specific etiology is suspected, testing should proceed with CMA and/or WES/WGS (and/or chromosome analysis in limited circumstances) and FXS testing. Particularly for global DD/ID, consider IEM testing, which has a low yield but high benefit for diagnosed patients.^,

As with DD/ID, the initial etiologic evaluation for ASD includes a detailed medical, developmental-behavioral, and family history; thorough physical and neurologic exam; and offer of a pediatric genetics referral.^, Certain disorders that have firmly established associations with ASD (eg, Angelman syndrome, Rett syndrome, FXS) may be identified through assessment followed by disorder-specific testing.^, If such a disorder is diagnosed and is consistent with the patient phenotype, then further testing to identify the etiology of ASD is not warranted. Otherwise, proceed with additional testing and/or consider referral for a medical genetics evaluation.

Chromosomal Microarray

CMA is a preferred first-tier test for DD/ID/ASD in patients for whom the causal diagnosis is unknown.^, CMA offers a higher diagnostic yield and detects more genetic abnormalities than many other techniques, especially in the presence of dysmorphic features and/or congenital anomalies; refer to the Comparison of Variants Detected by Different Methods of Genomic Tests table for additional detail.

If CMA results are normal, review of other first-tier test results and consideration of second-tier testing and/or referral to a medical geneticist are recommended. If CMA results are abnormal, genetic counseling is recommended.

FMR1 Testing for Fragile X Syndrome

FXS is the most common form of heritable ID and may be suggested by a family history of ID in male relatives. FXS is detected by testing for a cytosine-guanine-guanine (CGG) trinucleotide repeat expansion in the FMR1 gene that is not detectable by CMA or exome sequencing.^, The AAP recommends that all children who present with global DD/ID or ASD of unknown etiology be offered testing for FXS, and that referral for additional genetics testing should be provided if there is a consistent family history.^, The ACMG recommends FMR1 analysis for male children with ASD and for female children with a consistent family history and phenotype. Timely diagnosis supports access to early intervention measures and enables timely reproductive risk counseling. For additional information, refer to the Fragile X (FMR1)-Associated Disorders topic.

Metabolic and/or Mitochondrial Disorder Testing

Metabolic and/or mitochondrial disorder testing may be considered after reviewing newborn screening (NBS) results and assessing for clinical indicators that may suggest a metabolic disorder (eg, failure to thrive, unusual odors, hearing loss, and episodic symptoms). The AAP supports the consideration of screening for metabolic conditions in children who present with DD/ID/ASD.^, Many metabolic tests are available at a relatively low cost, and, despite the low prevalence of inherited metabolic conditions, the potential for improved outcomes after diagnosis and treatment is high.^,^,

Whole Exome Sequencing and Whole Genome Sequencing

WES and WGS are increasingly being used in clinical practice, and based on evidence of clinical value and detection rates, these methods are now strongly recommended for consideration as first- or second-line testing in the evaluation of pediatric-onset DD and ID. Individuals with clinical presentations suggestive of a chromosomal disorder, FXS, or Angelman syndrome/Prader-Willi syndrome should only receive WES or WGS after targeted testing has been performed. Similarly, individuals with a family history of a known disorder should first be tested for that condition. Rapid WES or WGS may be considered if acute clinical management is required. In individuals with ASD, the AAP states that WES may be performed as part of a medical genetics evaluation after negative CMA, fragile X, and MECP2 testing.

Both WES and WGS can identify a broader range of causative variants than other techniques, are well suited to identify rare variants, and may be particularly useful when used in conjunction with established methods such as CMA. The choice to pursue WES or WGS should be made based on clinical judgment and involve shared decision-making with patients and their families. Regardless of the technique chosen, informed consent is required, and genetic counseling is recommended before testing, including discussion of implications for family members and potential secondary findings.

Trio testing of the proband and both parents (if possible) is recommended for WES and WGS because it improves test performance—increasing the detection rate—by facilitating variant interpretation. This is especially true when rare variants are present.^, Evaluating additional family members may also be helpful. WES detects fewer variant types than WGS and therefore has a lower diagnostic yield; however, there are many considerations in test choice, which is why shared decision-making with a robust consent process is recommended.

Disorder-Specific Testing

MECP2

Second-tier testing for DD/ID after CMA, WES, or WGS includes complete MECP2 testing for affected female patients. MECP2 testing in these patients is indicated even in the absence of classic Rett syndrome features.

Second-tier testing for ASD, after CMA, includes full gene MECP2 sequencing (for Rett syndrome) in all female patients and MECP2 duplication testing in male patients with suggestive phenotypes.

PTEN

Patients of both sexes who have head circumferences >2.5 standard deviations (SDs) above the mean should have PTEN gene analysis if the ASD etiology is still unknown after initial evaluations.

Diagnostic Yields of Different Genetic Testing Methods

The diagnostic yield of different laboratory tests varies for D/D/ID and ASD.

Test	Approximate Diagnostic Yield in DD/ID	Approximate Diagnostic Yield in ASD
WGS	40%	—
WES	30%^,	Up to 20% Up to almost 30%
CMA	15-20%^,	10% Up to 25%
Fragile X testing	<5% (in individuals with mild to moderate DD/ID)	Up to 5%
MECP2 testing	<5% (in female patients with moderate to severe DD/ID)	<5% (female patients)
PTEN testing	—	5% (in individuals with head circumferences >2.5 SDs from the mean)
Karyotyping	<5% (excluding individuals with Down syndrome or other readily recognized chromosomal syndromes)^,	<5%
Metabolic or other disorder-specific testing not otherwise listed	Up to 10%	10%
Sources: Hyman, 2020; Schaefer, 2013; Manickam, 2021; Miller, 2010; Retterer, 2016; Tammimies, 2015; Michelson, 2011

Comparison of Variants Detected by Different Methods of Genomic Tests

Although traditional chromosome analysis, WES, and CMA assess variants across the genome, some variant categories may only be detected by a single test method.

Variants Detected by Different Genomic Testing Methods
Variant Category	Method
Variant Category	CMA	WES	WGS	Karyotype Analysis
Chromosomal CNVs (deletions/duplications)	Detected	May be detected^a	May be detected^a	Only large deletions and duplications ≥10-15 Mb
Sequence variants (eg, missense and point mutations)	Not detected	Detected	Detected	Not detected
Aneuploidy^b	Detected	May be detected^a,b	May be detected^a,b	Detected
Balanced chromosome rearrangements	Not detected	Not detected	May be detected^a	Detected
Unbalanced translocations	Detected^c	May be detected^a	May be detected^a	Detects unbalanced translocations ≥10-15 Mb
Trinucleotide repeat expansions	Not detected	Not detected	May be detected^a	Not detected
Aberrant methylation^d	Not detected	Not detected	Not detected	Not detected
Triploidy	Detected (with SNP analysis)	Not detected	Not detected	Detected
Consanguinity (regions of homozygosity)	Detected (with SNP analysis)	May be detected^a	May be detected^a	Not detected
Uniparental disomy	May be detected	May be detected^a	May be detected^a	Not detected
Exon-level deletions and duplications^e	May be detected	May be detected^a	May be detected^a	Not detected
Intergenic variants	Detected^f	Not detected	Detected	Detected if ≥10-15 Mb
Regions of homology	Detected (with SNP analysis)	May be detected^g	May be detected^g	Not detected
^aMay be included in testing and development of sequencing pipeline. ^bThe limit of detection of mosaicism varies depending on the size and type of genomic imbalance. For karyotype analysis, the detection of mosaicism depends on the number of cells/colonies counted. ^cCMA cannot provide structural (positional) information associated with genomic imbalance. ^dTesting that targets the specific disorder is required. ^eAdditional testing methods, such as MLPA, may be required. ^fSNP probes target unique sequence coding regions. Copy number probes provide coverage of noncoding regions, but variants in intergenic regions may not be interpreted and/or reported, depending on the size and genomic content. ^gThe limit of detection varies depending on the size of region, percent identity of homology, and probe coverage. CNV, copy number variant; Mb, megabase; MLPA, multiplex ligation-dependent probe amplification; SNP, single nucleotide polymorphism Sources: Manickam, 2021; Raca, 2022; Austin-Tse, 2022

ARUP Laboratory Tests

Panel Testing

2014314

Autism and Intellectual Disability Comprehensive Panel 2014314

Method

Tandem Mass Spectrometry/Electrophoresis/Spectrophotometry/ Gas Chromatography-Mass Spectrometry/Liquid Chromatography-Tandem Mass Spectrometry/Quantitative Liquid Chromatography-Tandem Mass Spectrometry, Genomic Microarray (Oligo-SNP Array), Polymerase Chain Reaction/Capillary Electrophoresis

Microarray

2003414

Cytogenomic SNP Microarray 2003414

Method

Genomic Microarray (Oligo-SNP Array)

2006267

Cytogenomic SNP Microarray Buccal Swab 2006267

Method

Genomic Microarray (Oligo-SNP Array)

Fragile X Testing

2009033

Fragile X (FMR1) with Reflex to Methylation Analysis 2009033